Despite costly attempts to reduce bacterial contamination of water, meat, and vegetables, Shiga toxin- producing E. coli (STEC) and related enteric pathogens (e.g. Salmonella, Shigella, Yersinia) are causing increasingly frequent outbreaks of food-borne diarrheal disease, thus constituting enormous health burdens. These pathogens use a type III secretion system (T3SS) to inject virulence proteins (effectors) into host cells. While T3SS effectors clearly play essential roles in bacterial virulence, the mechanisms by which they subvert the functions of host cells to promote pathogen survival are incompletely characterized. We are studying the mammalian signal transduction pathways targeted by STEC effectors, with the ultimate goal of improving our ability to prevent and to treat bacterial infections. We have made the important and unique discovery that the STEC strains associated with severe diarrheal disease outbreaks in humans express a pair of homologous effectors that differentially regulate host innate immunity by disrupting the transcriptional responses to infection that are normally coordinated by the NF-kB pathway. NF-kB activity at key innate immune response genes is regulated by ribosomal protein S3 (RPS3), which possesses an accessory nuclear function as an NF-kB subunit. We discovered that the NleH1 effector protein inhibits RPS3 nuclear translocation, reducing NF-kB activity at specific promoters. Specifically, NleH1 inhibits the Ik kinase complex (IKK) from phosphorylating RPS3, a critical requirement for its nuclear translocation. STEC strains also encode a homologous effector named NleH2. Despite sharing 84 % identity with NleH1, NleH2 stimulates rather than inhibits RPS3/NF-kB-dependent transcription. We hypothesize that the NleH1 and NleH2 effectors promote bacterial virulence by subverting the pro- inflammatory responses to infection normally regulated by RPS3/NF-kB and propose the following specific aims: 1) Quantify the importance of NleH effectors to bacterial virulence using animal models of diarrheal disease. 2) Characterize the molecular mechanism by which NleH1 inhibits IKK phosphorylation of RPS3 to prevent its nuclear import. 3) Elucidate mechanistic differences between NleH1 and NleH2 and their pathophysiological significance in regulating RPS3/NF-kB-dependent signaling. Successful completion of the proposed research will 1) reveal how these bacterial effectors selectively modulate innate immunity; 2) clarify how pathogens have evolved to co-opt the accessory nuclear functions of ribosomal proteins; and 3) characterize how bacteria have integrated their virulence proteins into host signal transduction pathways in specific spatiotemporal contexts.